The focus of this proposal is on the role of DNA methylation in the regulation of pyelonephritis-associated pili (pap) gene expression in uropathogenic Escherichia coli. Pap pili expression is subject to a phase variation regulatory mechanism which involves DNA methylation and specific methylation inhibition at two GATC sites (GATC1028 and GATC1130) within pap regulatory DNA sequences. This mechanism appears to be different from other phase variation systems previously studied. The overall objective of this proposal is to determine the mechanism by which the methylation of the GATC1028 and GATC1130 sites is inhibited by the papI and methylation blocking factor (mbf) gene products, and how these gene products regulate pilin transcription. These proteins will be used to analyze protein-DNA interactions in vitro using DNaseI and methylation protection assays. To determine the protein-DNA interactions occurring in transcriptionally inactive (off) and active (on) cells, we will use in vivo DNA footprinting. An abortive transcriptional initiation assay will be developed to determine if MBF, PapI and cAMP-CAP are sufficient to stimulate pap transcription by RNA polymerase. If they are not, we will use this assay to purify additional factors from crude E. coli extracts. The role(s) of Dam methylase in pap gene regulation will be examined by determining the effects of in vitro methylation on protein-DNA interactions and transcription initiation. In addition to the above proposed biochemical approach, pap regulatory mutants will be isolated after random mutagenesis. In these studies, pilin transcription will be analyzed using single copy pap-lac gene fusion constructs. A search for both pap-linked and unlinked second site revertants will be carried out as well. Regulatory mutants will be characterized by DNA sequence analysis and by DNA-protein interaction analysis as described above. Site-directed mutagenesis will be used to further probe the functions of specific pap regulatory sequences that are identified by random mutagenesis. The experiments proposed are important and significant because they explore a new area in bacterial gene regulation; the epigenetic modulation of gene expression by DNA methylation inhibition.